The long-term objective of this research is to clarify the genetic mechanisms by which hematopoietic stem cells proliferate and differentiate into mature blood cells. More specifically, this project will focus on the Runxl transcription factor in hematopoietic stem cell function. Runxl is a DNA binding transcription factor. However, its critical target genes in hematopoiesis are not known. Chromosomal translocations and mutations of Runxl are involved in the pathogenesis of leukemia and myelodysplastic syndrome. Runxl gene dysruption in mice causes embryonic lethality due to complete lack of definitive hematopoiesis. Conditional knockout of this gene in adult life causes perturbation of normal proliferation and differentiation in various hematopoietic lineages. This proposal will test a hypothesis that Runxl directly regulates the expression of a group of critical genes involved in the maintenance and differentiation of hematopoietic stem cells. This project will proceed in three steps. In Specific Aim 1, the mouse multipotent hematopoietic stem cell line EML, which is a good in vitro model of normal hematopoiesis, will be used for ChIP on chip analysis. ChIP on chip analysis combines chromatin immunoprecipitation and DNA tiling microarray analysis, and is a powerful technique for genome-wide search for transcription factor target genes. In Specific Aim 2, expression microarray analysis will be performed on hematopoietic stem cells from the Runxl conditional knockout mouse. The transcriptional profile of these cells will reveal the genes whose expression is controlled by RUNX1. The role of Runxl in illness will then be approached throuh the transcriptional profile of the Runxl (Ala224fsTer228) mutation, found in a patient with MDS/AML. In Specific Aim 3, the data obtained in Specific Aims 1 and 2 will be combined to picture the network of genes that are under direct control of the Runxl transcription factor. The major target genes and the downstream pathways affected by this gene will be characterized focusing on hematopoietic stem cell function. The most significant genes will be selected and tested for their significance in the hematopoietic system, using both in vitro and in vivo approaches. Normal hematopoiesis is a result of controlled expression of various genes in the hematopoietic stem cell. Runxl is a master regulator of gene expression in hematopoietic stem cells. Despite intense research in the field, the most critical Runxl target genes for normal hematopoiesis is still unknown. The goal of this project is to discover the genes indispensable for normal hematopoiesis. Understanding of the precise molecular mechanisms that control hematopoiesis is essential for the development of better therapeutic drugs for hematopoietic disorders.